Summary: Project 3 DeGrado An expanding body of evidence argues that besides PrP, A, tau and synuclein can become prions and cause neurodegeneration. We seek to understand the assembly of prion precursors into oligomers and fibers, and to correlate the formation of specific conformational forms with toxicity and transmissibility. The small size of A makes it an attractive molecule to study with respect to conversion into prions that are presumably oligomers. We propose to determine the steps required for A to acquire a self-propagating conformation that templates the assembly of additional monomers into oligomers and possibly fibers. The molecular structures of the key intermediates in this process are poorly understood, and it is not known whether the transmissible particle has the parallel cross-beta structure seen in fibers. While it is generally accepted that A oligomers rather than fibers represent the toxic species, the relationship between toxicity and the size, conformation, and dynamics of the oligomers is unknown. We will develop new chemical tools to probe and dissect the steps of aggregation and amyloid formation. We will develop novel chemical strategies to rapidly trigger A association and follow the evolution of its conformation over time. We also will design peptide mimetics and foldamers that enhance or inhibit assembly by stabilizing specific intermediates, allowing one to obtain better defined populations for building correlations between structure, transmissibility and toxicity. Finally, we will develop crosslinking strategies that can be used to obtain a footprint of the conformational ensemble of synthetic versus natural A, PrP, and tau at various levels of assembly.